Project Summary In order to complement the current sputum based methods of tuberculosis (TB) diagnosis and treatment monitoring, novel assays are required to detect the presence of live Mycobacterium tuberculosis (Mtb) bacilli in patients. The discovery of Mtb-specific protein encapsulation in exosomes released from Mtb-infected macrophage culture was the impetus for exploration of the phenomenon in the exosome fraction of serum; targeted mass spectrometry was applied to confirm the identification of over a dozen Mtb-antigens in exosomes isolated from sputum-confirmed tuberculosis patients. Exosomes are small vesicles released from nucleated cells and are a rich-source of biomarkers. To complement and extent the utility of serum exosomes as markers of treatment efficacy, this proposal focuses on the analysis of ultrapure exosomes using unbiased mass spectrometry (MS) for the discovery of Mtb proteins and their kinetic response in 25 TB patients over 4 points during anti-mycobacterial treatment. Targeted MS assays (MRM) will be used to confirm the proteomic differences. Statistical modeling will be used to determine a proteomic signature consistent with clearance of Mtb from the sputum and will be performed with a training set (n = 256, baseline/treatment pairs) and a validation set (n = 64, baseline/treatment pairs) of serum exosome samples. Ultimately, an Mtb protein signature in serum exosomes can be used in conjunction with sputum diagnostics or when sputum is not available (in children and during treatment when production/cough resolves) and will reflect the elimination of transmissible bacilli in the sputum. In order to translate discoveries to a monitoring assay relevant to treatment monitoring in the clinic our methods must be simplified to a platform independent of mass spectrometry, however for the purpose of pre- clinical animal research MRM-MS remains a feasible option for novel drug and treatment regimen efficacy trials.